We propose to continue studies directed towards understanding the mechanisms by which Drosophila alcohol dehydrogenase (Adh) genes are regulated during development. The cis-acting DNA sequences required for normal temporal and tissue specific Adh gene expression will be identified by introducing mutations into cloned Adh genes and then studying the effects of these alterations on gene expression in vivo. Expression of the mutant genes will be analyzed by introducing them into the D. melanogaster germ line by P-element transformation. The sequence requirements for the switch in Adh promoter utilization during development in D. melanogaster will also be determined in a similar manner. We also plan to continue a parallel study of the regulation Adh genes in D. mulleri, a Drosophila species win which two genes rather than two promoters are differentially regulated during development. We have recently isolated and sequenced the D. mulleri genes, and have shown that they are appropriately regulated when introduced into the D. melanogaster germ line by P-element transformation. A comparison of the cis-acting regulatory sequences in the two Drosophila species should provide significant new information regarding the mechanism of Adh promoter switching. Finally, we plan to pursue a variety of approaches to identify and clone the genes that encode trans-acting factors that regulate Adh gene expression. These approaches include in vitro DNA binding studies, a genetic screen in yeast for genes encoding proteins that bind to Adh regulatory sequences, and a Xenopus oocyte microinjection assay for Adh regulatory factors. In addition, we plan to screen for Adh regulatory mutants in Drosophila. If trans-acting Adh regulatory genes are cloned by one or more of these approaches, the cloned genes will be characterized and used to produce sufficient amounts of protein for detailed in vitro binding and transcription studies.